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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Mol Carcinog. 2022 Oct 26;62(2):236–248. doi: 10.1002/mc.23479

Sulforaphane inhibits CD44v6/YAP1/TEAD signaling to suppress the cancer phenotype

Xi Chen 1, Gautam Adhikary 1, Emily Ma 1, John J Newland 2, Warren Naselsky 2, Wen Xu 1, Richard L Eckert 1,3
PMCID: PMC9851963  NIHMSID: NIHMS1842453  PMID: 36285644

Abstract

Sulforaphane (SFN) is a promising cancer prevention and treatment agent that strongly suppresses the cutaneous squamous cell carcinoma (CSCC) cell cancer phenotype. We previously showed that YAP1/TEAD signaling is a key pro-cancer stimulator of the aggressive CSCC cell cancer phenotype. However, SFN-responsive upstream regulators of YAP1/TEAD signaling are not well characterized and so there is a pressing need to identify these factors. We show that CD44v6 knockdown reduces YAP1/TEAD-dependent transcription and target gene expression, and that this is associated with reduced spheroid formation, invasion and migration. CD44v6 knockout cell lines also display reduced YAP1/TEAD activity and target gene expression and attenuated spheroid formation, invasion, migration and tumor formation. An important finding is that SFN treatment suppresses CD44v6 level leading to a reduction in YAP1/TEAD signaling and marker gene expression. Sox2 level and EMT are also reduced. Forced expression of constitutive active YAP1 in CD44v6 knockdown cells partially restores the aggressive cancer phenotype. These important findings suggest that CD44v6 drives YAP1/TEAD signaling to enhance the CSCC cell cancer phenotype and that SFN treatment reduces CD44v6 level/function which, in turn, reduces YAP1/TEAD signaling leading to reduced stemness, EMT and tumor growth.

Keywords: Sulforaphane, squamous cell carcinoma, cancer prevention, CD44v6, YAP1/TEAD signaling

Introduction

Cutaneous squamous cell carcinoma (CSCC) is one of the most prevalent human cancers1,2. CSCC is commonly treated by surgical resection; however, a significant number of resected cancers relapse to form aggressive chemotherapy resistant cancer. Our previous studies characterized epidermal cancer stem-like cells (ECS cells), which comprise 0.15% of tumor cells, and exhibit enhanced proliferation and invasion as compared to non-stem cancer cells3. Injection of 100 ECS cells is sufficient to drive robust tumor growth in immune-compromised mice3. Therefore, it is important to identify cell survival factors that are enriched in ECS cells and can be targeted to suppress cancer growth and recurrence.

Sulforaphane (SFN) is a natural occurring compound, which is known to have anti-cancer action in multiple cancers, including skin cancer4–11. SFN treatment increases reactive oxygen species (ROS), inhibits histone deacetylase (HDAC) activity, promotes cell apoptosis, and induces cell cycle arrest in cancer cells12,13. We have shown that SFN suppresses YAP1/ΔNp63α signaling14, induces p21Cip1 15, interacts with transglutaminase 28, suppresses PRMT5/MEP50 signaling9 and suppresses the ECS cell cancer phenotype and tumor formation11.

CD44 is a recognized cancer stem cell marker which is upregulated in a host of cancer types16. CD44 variants are generated by alternative splicing and have different functions in tissues and tumors16. For instance, some CD44 variants serve as co-receptors for growth factors that promote cell signaling17,18. CD44 expression is associated with a highly proliferative, invasion, and metastatic cancer phenotype16. In the present study, we focus on the CD44v6 variant which is an important pro-cancer isoform expressed in epidermal keratinocytes and CSCC cells17,19.

Hippo signaling plays a central role in controlling organ growth during development20,21 and yes-associated protein 1 (YAP1) is an important effector of the Hippo pathway. In the absence of LATS1 activity, a key regulatory kinase in the Hippo signaling cascade, non-phosphorylated YAP1 accumulates in the nucleus to stimulate cell proliferation. In contrast, activation of LATS1 signaling phosphorylates YAP1 leading to its sequestration in the cytosol and degradation leading to reduced cell proliferation22,23. YAP1 is often activated and localized in the nucleus in tumor cells where it interacts with TEAD DNA transcription factors to initiate transcription of genes including connective tissue growth factor (CTFG), cysteine-rich angiogenic inducer 61 (CYR61), cyclin D1, and others24. In CSCC, YAP1/TEAD target genes include CYR61, CCND1, CTGF and the pro-cancer collagen genes, COL1A2 and COL3A125. However, it is increasingly apparent that multiple mechanisms control YAP1/TEAD function26–28 and recent studies show that CD44 can regulate YAP1 signaling and YAP1 target gene expression in several cancer types29–31.

Our goal is to identify upstream regulators of YAP1/TEAD transcription that can be targeted to reduce YAP1/TEAD signaling in CSCC. In the present study, we show that SFN reduces CD44v6 level and that this is associated with reduced YAP1 level and reduced spheroid formation, invasion, and migration. To confirm a role of CD44v6 as a mediator of SFN suppression of YAP1/TEAD signaling, we show that the CD44v6 knockdown or knockout reduces YAP1/TEAD transcription and target gene expression and attenuates the cancer phenotype and tumor formation. These findings suggest that CD44v6 maintains YAP1/TEAD signaling in CSCC cells and that SFN suppression of CD44v6 function leads to a reduction in YAP1/TEAD signaling to suppress the CSCC cancer phenotype and tumor formation.

Materials and Methods

Antibodies and reagents

DMEM (11960-077), L-Glutamine (25030-164), 0.25% trypsin-EDTA (25200-056), and sodium pyruvate (11360-070) were purchased from Gibco (Grand Island, NY). DMEM/F12 (1:1) medium (DMT-10-090-CV) was purchased from Mediatech INC (Manassa, VA). B27 serum-free supplement (17504-044), Superscript III reverse transcriptase (18080051) was obtained from Invitrogen (Frederick, MD). Sulforaphane (S8044) was purchased from LKT Laboratories Inc (St. Paul, MN). CD44v6 (ab78960), Sox2 (ab15830-100), Twist (ab49254), and Slug (ab27568) antibodies were purchased from Abcam (Cambridge, MA). YAP1 (4912), YAP1-P (13008), LATS1 (9513), LATS1-P (8654), E-Cadherin (3195S), CCND1 (55506), CRY61 (14479) and pan-TEAD (13295) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Transglutaminase 2 (MAB3839) and β-actin (A5441) antibodies, DAPI (D9542), bovine serum albumin (B4287), epidermal growth factor (EGF) (E4269), insulin (19278), and heat-inactivated fetal calf serum (FCS) were purchased from Sigma-Aldrich (St. Louis, MO). Matrigel (354234) and BD BioCoat Millicell inserts (d = 1 cm, 8 mM pore size, #353097) were obtained from BD Bioscience (Frankin Lakes, NJ). Peroxidase-conjugated sheep anti-mouse IgG (NXA931) and Peroxidase-conjugated donkey anti-rabbit IgG (NA934V) antibodies were obtained from GE Healthcare (Laurel, MD). YAP1-siRNA (S102662954) was purchased from Qiagen (Valencia, CA). CD44v6-siRNA (SMARTpool, M-009999-03-0005) was purchased from Dharmacon (Lafayyette, CO). Control-siRNA (sc-37007) were purchased from Santa Cruz (Dallas, TX). The YAP1/TEAD inhibitor, CA3 (CIL56), was purchased from Selleckchem. Plasmids, including pGL3basic (128046), pcDNA3.1 (V79020), pcDNA3-FLAG-YAP(S127A) (27370), 8xGTIIC-luciferase (34615) and px459V2.0_Conc2 (134451), were purchased from Addgene (Watertown, MA). FuGENE 6 Transfection Reagent (E2312) and luciferase assay substrate (E1910) were purchased from Promega (Madison, WI). Light Cycler 480 SYBR Green I Master mix (04707516001) was purchased from Roche Diagnostics (Florham Park, NJ). The IIIustra RNAspin mini kit (25-0500-71) was purchased from GE Healthcare (Laurel, MD). Two-tailed Student’s t-test was used for binary comparison between control and experimental groups. All experiments were repeated three times and the data is presented as mean ± SEM32.

Immunoblot

Cells and tissues extracts were prepared in Laemmli buffer (0.063 M Tris-HCI, pH 7.5, 10% glycerol, 5% SDS, 5% β-mercaptoethanol). Equivalent amounts of proteins were electrophoresed on 8% – 12% SDS-PAGE gels and the separated proteins were transferred onto nitrocellulose membranes which were then blocked in the 5% non-fat milk for 1 h, before they were incubated overnight with primary antibody. The membranes were washed and then incubated with secondary antibody for 2 h before antibody binding was visualized using ECL (Amersham) chemiluminescence detection technology.

Cell Culture

SCC-13 cells33 are aggressive tumor-forming epidermal squamous carcinoma cells and HaCaT cells34 are epidermis-derived immortalized but non-tumorigenic cells. SCC-13 and HaCaT monolayer cultures are routinely grown in culture medium supplemented with 10% FCS3. Enriched epidermal cancer stem-like cells (ECS cells) were used in all experiments3,35. The ECS cells were derived by growth of cells as unattached spheroids in DMEM/F12 (1:1) medium containing 2% B27 serum-free supplement, 20 ng/ml EGF, 0.4% bovine serum albumin and 4 μg/ml insulin. The resulting spheroids are dissociated into single cells and used for proliferation, invasion, migration and tumor growth experiments. Spheroid enrichment increases the ECS cell population from 0.15% (monolayer cultures) to 15 – 20% of the total cell population. Enriched ECS cells are used in these experiments because this population is more aggressive in proliferation, spheroid formation, invasion, migration and tumor formation3. For electroporation, cells were electroporated with 3 μg of siRNA or plasmid using the T-018 setting on the AMAXA Electroporator. The cells were recovered for 48 h, and were then harvested for use in spheroid formation, invasion, and migration assays.

SCC-13 cell derived CD44v6-knockout cell clones, SCC13-CD44v6-KOc1-2-3 and SCC13-CD44v6-KOc1-2-2, were generated by using CRISPR/Cas9 technology. CD44v6-specific guide RNA (5’-caccGCTGTGCAGCAAACAACACAGGGG) and reverse (5’-aaacCCCCTGTGTTGTTTGCTGCACAGC) strands were identified at http://crisper.technology and cloned in the U6-driven pSpCas9(BB)-2A-Puro (PX459) V2.0 vector. The vector was then electroporated into SCC-13 cells using the AMAXA electroporator and cell clones were selected by treatment with 2 μg/ml puromycin. Single cell clones were selected by dilution cloning and the absence of CD44v6 was confirmed by immunoblot.

Spheroid formation, invasion and migration assays

Cells were seeded into 6 well Ultra-Low attachment plates at a density of 40,000 per well. The cells were cultured in stem cell medium and spheroid growth was monitored from 0 – 3 days. Images were produced and spheroid size and number were determined by Image J analysis. For invasion assay, BD BioCoat Millicell inserts were pre-coated with 250 μg/ml Matrigel. Enriched ECS cells (25,000 cells/well) were seeded atop the Matrigel layer in medium containing 1% FCS and cell invasion into the lower chamber containing medium supplemented with 10% FCS was monitored at 20 h by DAPI staining. For migration assay, enriched ECS cells were grown as a confluent monolayer and then wounded with a 10 μl tip. Wound closure was monitored from 0 – 24 h.

qRT-PCR

Total RNA was extracted using the IIIustra RNAspin mini kit and 1 μg of total RNA was reverse transcribed to cDNA using Superscript III reverse transcriptase. mRNA level was measured using the light Cycler 480 SYBR Green I Master mix. The primers include: cyclophilin A (forward: 5’-CAT CTG CAC TGC CAA GAC TGA, reverse: 5’-TTC ATG CCT TCT TTC ACT TTGC), CCND1 (forward: 5′-TGA AGG AGA CCA TCC CCC TG-3ʹ, reverse: 5′-TGT TCA ATG AAA TCG TGC GG-3ʹ), CYR61 (forward: 5′-CAC ACC AAG GGG CTG GAA TG-3ʹ, reverse: 5′-CCC GTT TTG GTA GAT TCT GG-3ʹ) and CTGF (forward: 5′-GGA AAT GCT GCG AGG AGT GG-3ʹ, reverse: 5′-GAA CAG GCG CTC CAC TCT GTG-3ʹ)25.

Luciferase assay

Cells were electroporated with 3 μg of the appropriate siRNA and permitted to recover for 48 h before addition of 1 μg pGL3basic or the pGL3-B-8xGTIIC-luciferase TEAD reporter plasmid delivered by transfection using Fugene 6. At 24 h the cells were harvested for the detection of TEAD promoter activity.

Tumor xenograft model

Single cell suspensions from SCC-13 wild-type and SCC13 CD44v6 knockout cells were grown as spheroids to select enriched ECS cells which were then suspended in PBS containing 30% Matrigel for subcutaneously injected of 0.1 million cells into each front flank in immune compromised NSG (NOD/SCID/IL2Rg−/−) mice. Each group included 5 mice and tumor growth was monitored from 0 – 4 weeks by measuring tumor diameter3,36. Tumor sizes were calculated by the formula Volume = 4/3π × (diameter/2)3. At the end of the experiment, tumor tissue was collected and processed for extract preparation. The animal studies were reviewed and approved by the institutional Animal Care and Use Committee (IACUC) and followed the standard international practices for treatment of animals.

Results

Sulforaphane suppresses the cancer phenotype

Enriched ECS cells, derived by culturing monolayer cells as unattached spheroids in stem cell selection medium3, were used in all experiments. This enriches the ECS cell population from 0.15% (monolayer cultures) to 15 – 20% of the total cell population. Enriched ECS cells are used because they are more aggressive in proliferation, spheroid formation, invasion, migration and tumor formation3.

We have previously shown that SFN treatment reduces YAP1/ΔNp63α signaling to reduce the cancer phenotype14. Our present data confirm these findings. We show that SFN treatment reduces SCC-13 spheroid formation (Fig. 1A). A partial reduction in spheroid diameter is observed following treatment with 10 μM SFN and a further reduction in the presence of 20 μM SFN. Based on these finding we have used 20 μM SFN in subsequent experiments. SFN treatment also reduced invasion through Matrigel (Fig. 1B), and migration on plastic (Fig. 1C), and similar reductions are observed for HaCaT cells (Fig. 1D/E/F). Of particular interest, we found that SFN treatment was associated with a reduction in CD44v6 and YAP1 levels (Fig. 1G). Moreover, the loss of YAP1 was associated with a reduction in YAP1/TEAD target gene mRNA levels in both SCC-13 and HaCaT cells (Fig. 1H). Because CD44v6 is an important stimulator of cancer cell stemness and survival, and has been shown to regulate YAP1/TEAD signaling in some cancers29–31, we examined its role in greater detail.

Fig. 1. SFN suppresses the CSCC cell cancer phenotype.

Fig. 1

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A/B/C SFN reduces SCC-13 cell spheroid formation, invasion, and migration. D/E/F SFN reduces HaCaT cell spheroid formation, invasion, and migration. G The reduced cancer phenotype is associated with reductions in CD44v6, YAP1, and YAP1-P. H mRNA levels of YAP1 target genes, including CCND1, CYR61, and CTGF, are decreased in SFN treated cells. The single asterisk indicates a significant reduction, n = 3, p < 0.001.

CD44v6 maintains the cancer phenotype and YAP1 activity

The novel observation that CD44v6 is reduced in SFN treated cells suggests that it may mediate SFN action. To understand the role of CD44v6 we first performed knockdown experiments. We show that CD44v6-siRNA treatment reduces SCC-13 cell spheroid formation (Fig. 2A), invasion (Fig. 2B), and migration (Fig. 2C), which phenocopies the responses to SFN. Moreover, CD44v6 knockdown is associated with reduced total YAP1 and YAP1-P levels (Fig. 2D). CD44v6 knockdown produced similar changes in HaCaT cells, including a reduction in spheroid formation (Fig. 2E), invasion (Fig. 2F) and migration (Fig. 2G), and suppressed total and phosphorylated YAP1 levels (Fig. 2H). This suggests a role for CD44v6 acts to maintaining YAP1-related signaling in both cell types.

Fig. 2. CD44v6 maintains the cancer phenotype and YAP1 levels.

Fig. 2

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A/B/C Targeted CD44v6-siRNA treatment reduces SCC-13 cell spheroid formation, invasion, and migration. D Immunoblot confirms CD44v6 knockdown and associated reduction in YAP1 and YAP1-P. E/F/G CD44v6-siRNA treatment reduces HaCaT cell spheroid formation, invasion, and migration. H CD44v6 knockdown HaCaT cells display reduced YAP1 and YAP1-P levels. I Control- and CD44v6-siRNA treated cells were transfected with EV-Luc or 8xTllC-luc vector. CD44v6 knockdown reduces YAP1/TEAD transcriptional activity in both cell types. J CD44v6-siRNA treatment reduces the mRNA abundance of the YAP1 target genes, CYR61 and CCND1. The single asterisk indicates a significant decrease, n = 3, p < 0.001.

We therefore further examined the impact of CD44v6 knockdown on YAP1/TEAD-dependent transcription and YAP1/TEAD marker gene expression. To measure the impact on YAP1/TEAD-dependent transcription, SCC-13 and HaCaT cells were electroporated with Control (EV-Luc) or TEAD response element (8xTIIC-Luc) encoding reporter plasmids in the presence of Control or CD44v6-specific siRNA. Fig. 2I shows that activity of the control vector is not modulated by CD44v6 knockdown, but that there is a dramatic reduction in YAP1/TEAD response element vector activity in CD44v6 knockdown cells. The reduction in transcriptional activity following CD44v6 knockdown is correlated with reduced YAP1/TEAD target gene (CCND1, CYR61) mRNA (Fig. 2J). In addition, CTGF mRNA levels trended lower in CD44v6 knockdown cells, but the reduction was not statistically significant.

The role of YAP1 in maintaining the cancer phenotype

We next assessed the impact of YAP1 knockdown. These findings show that YAP1 loss reduces spheroid growth (Fig. 3A) and cell invasion (Fig. 3B), and that this is associated with a reduction in CYR61 and cyclin D1 levels (Fig. 3C). We also treated with CA3, an inhibitor of YAP1/TEAD signaling37 and show that this agent reduces spheroid growth (Fig. 3D) and cell invasion (Fig. 3E), and also reduces CYR61 and cyclin D1 levels (Fig. 3F). YAP1 knockdown (Fig. 3G/H/I) and CA3 treatment (Fig. 3J/K/L) produced similar reductions in HaCaT cells. These findings confirm that YAP1 knockdown and YAP1 inhibitor treatment mimic the reduction in YAP1/TEAD target gene levels observed in CD44v6 knockdown cells.

Fig. 3. YAP1 signaling is required to maintain the aggressive cancer phenotype.

Fig. 3

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Cells were treated with Control- or CD44v6-siRNA, or the YAP1/TEAD inhibitor, CA3. A/B YAP1 knockdown reduces SCC-13 cell spheroid formation and invasion. C Immunoblot confirms a reduction in YAP1 and YAP1-P, and YAP1 targets in YAP1-siRNA treated cells. D/E CA3 treatment reduces SCC-13 cell spheroid formation and invasion. F CA3 treatment reduces YAP1, increases YAP1-P, and reduces CYR61 and Cyclin D1. G/H YAP1-siRNA reduces HaCaT cell spheroid formation and invasion. I YAP1 knockdown reduces YAP1 and YAP1-P, and CYR61 and Cyclin D1. J/K CA3 treatment reduces HaCaT cell spheroid formation and invasion. L CA3 treatment reduces YAP1, increases YAP1-P, and decreases the YAP1 target (CYR61 and Cyclin D1) levels. The single asterisk indicates a significant decrease, n = 3, p < 0.001.

Constitutively active YAP1 restores the cancer phenotype

Our findings show that the cancer phenotype is attenuated in CD44v6 knockdown cells and that this is associated with reduced YAP1 function. To determine if YAP1 loss is required for attenuation of the phenotype, we expressed constitutively active YAP1 in CD44v6 deficient cells to test if this restores the cancer phenotype. We show that CD44v6 knockdown reduces SCC-13 cell spheroid formation (Fig. 4A), invasion (Fig. 4B) and migration (Fig. 4C) and that vector mediated expression of YAP(S127A) partially restores spheroid formation, invasion and migration. Fig. 4D/E/F show that spheroid formation, invasion and migration are also reduced in HaCaT cells, and that YAP(S127A) expression partially restores the aggressive cancer phenotype. The immunoblots shown in Fig. 4G/H confirm a reduction in YAP1 level in CD44v6-siRNA treated cells and elevated YAP1 levels in cells treated with constitutively active YAP1 expression vector.

Fig. 4. CD44v6 actions are mediated via YAP1 signaling.

Fig. 4

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Cells were treated with Control- or CD44v6-siRNA in the presence or absence of YAP(S127A). CD44v6-siRNA treatment reduces SCC-13 cell spheroid formation, invasion and migration and these responses are partially antagonized by constitutively active YAP1. D/E/F CD44v6-siRNA treatment reduces HaCaT cell spheroid formation, invasion and migration and these responses are partially antagonized by constitutively active YAP1. The single asterisk indicates a significant decrease, n = 3, p < 0.001, compared to the control, and double asterisks indicate a significant increase, n = 3, p < 0.005 compared to the CD44v6-siRNA groups. G/H The immunoblots show the levels of CD44v6 and YAP1 in each treatment group.

Characterization of CD44v6 knockout cells

To further demonstrate a role for CD44v6, we derived five clonal CD44v6 knockout cell lines from SCC-13 cells. As anticipated, the CD44v6 knockout cell lines display reduced spheroid formation (Fig. 5A), invasion (Fig. 5B) and migration (Fig. 5C). Fig. 5D shows that CD44v6 knockout is associated with reduced activity of 8xTIIC-Luc, the YAP1/TEAD transcription reporter plasmid, which indicates a reduction in transcription. Moreover, this reduction in transcription is associated with reduced YAP1 and increased YAP1-P levels, and reduced YAP1/TEAD target protein (CYR61, Cyclin D1) levels. The level of the Sox2 stem cell marker is also suppressed. Moreover, EMT marker changes, including reduced Twist in all CD44v6 knockout cell lines and increased E-Cadherin levels in a subset of these lines, suggests that EMT is attenuated.

Fig. 5. CD44v6 knockout cells display an attenuated cancer phenotype.

Fig. 5

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Five SCC-13 CD44v6 knockout clones were generated using CRISPER/Cas9 technology. A/B/C CD44v6 knockout cells display reduced spheroid formation, invasion, and migration. D Wild-type and CD44v6 knockout cells were transfected with EV-Luc and 8xTIIC-Luc YAP1/TEAD transcriptional reporter plasmids. CD44v6 knockout reduces YAP1/TEAD transcriptional activity. E CD44v6 knockout reduces YAP1, increases YAP1-P, reduces TEAD, and decreases the YAP1 targets, CYR61 and Cyclin D1. In addition, Sox2 is reduced and EMT is reduced (decreased fibronectin and Twist, increased E-cadherin). Slug levels do not change. The single asterisk indicates a significant decrease, n = 3, p < 0.001.

SFN treatment reduces CD44v6 level in tumors

We next examined the impact of SFN on CD44v6 level in tumors. We subcutaneously injected 0.1 million wild-type SCC-13 ECS cells into the front flanks of NSG mice and then initiated treatment with SFN. Fig. 6A/B shows that SFN treatment reduces tumor growth and that this is associated with reduced CD44v6 level. Fig. 6B also shows a reduction in YAP1, YAP1-P, pan-TEAD, and YAP1/TEAD target protein (cyclin D1 and CYR61) levels. EMT is also reduced as evidenced by reduced Twist and Slug and increased E-Cadherin. These findings suggest that SFN treatment reduces CD44v6 expression, YAP1/TEAD-dependent events, and reduces stemness and EMT.

Fig. 6. CD44v6 knockout mimics SFN action on tumor growth and signaling.

Fig. 6

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A/B SCC-13 spheroid-derived ECS cells (0.1 million) were subcutaneously injected into each front flank of five NSG mice (n = 10 tumors/treatment group) followed by treatment with SFN delivered by gavage three times per week for four weeks. SFN treatment reduces tumor formation, and this is associated with the reduced YAP1 and TEAD, increased YAP1-P, reduced CYR61 and Cyclin D1, and a reduced EMT. C/D Wild-type and CD44v6 knockout cell-derived ECS cells (0.1 million) were subcutaneously injected into each front flank of five NSG mice (n = 10 tumors/treatment group) and tumor growth was monitored for 4 weeks. E YAP1, pan-TEAD, cyclin D1 and CYR61 are reduced in CD44v6 knockout tumors. Sox2 is reduced and EMT is attenuated. The single asterisk indicates a significant decrease, n = 3, p < 0.001.

CD44v6 knockout impact on tumor formation

To specifically confirm a role for CD44v6 in tumors, we subcutaneously injected 0.1 million wild-type and CD44v6 knockout (SCC13-CD44v6-KOc1-2-2 and SCC13-CD44v6-KOc1-2-3) cells into the front flanks of NSG mice and monitored the effects on tumor growth and marker protein level. The CD44v6 knockout clones produced tumors that grow extremely slowly (Fig. 6C/D) and display reduced YAP1, elevated YAP1-P, and reduced pan-TEAD, cyclin D1 and CYR61 levels (Fig. 6E) indicating a reduction in YAP1/TEAD signaling. In addition, the Sox2 cancer stem cell marker is reduced and EMT is reduced as evidenced by reduced fibronectin and Twist, and increased E-cadherin. Slug levels, in contrast, are not changed (Fig. 6E).

Discussion

SFN treatment suppresses YAP1/TEAD signaling

SFN is an isothiocyanate that is present at abundant levels in cruciferous vegetables including broccoli13,38,39. It is a promising anti-cancer agent that is highly effective in suppressing tumor growth in preclinical models in a wide range of cancers8,9,11,14,22,26,40–42. YAP1/TEAD signaling is a centrally important skin cancer cell survival mechanism14,25 and we have shown that SFN treatment reduces YAP1/TEAD function to suppress the skin cancer phenotype and tumor formation14. We are interested in identifying upstream regulators that may mediate SFN suppression of YAP1/TEAD function.

CD44v6 in epidermal skin squamous cell carcinoma

CD44 is an important cancer stem cell marker that is spliced as different variants in a cell type-dependent manner16 and CD44 dysregulation is frequently observed in advanced cancer where it is positively correlated with a poor patient prognosis43. CD44v6 is an important CD44 isoform that is present in human epidermal keratinocytes19,44,45 and is increased during carcinogenesis19. CD44v6 acts to maintain stemness and an aggressive cancer phenotype and is an important anti-cancer target.

Our present studies shows that SFN treatment reduces CD44v6 level and that this is associated with reduced YAP1/TEAD signaling and reduced spheroid formation, invasion and migration. Furthermore, we show that CD44v6 knockdown produces a similar reduction in YAP1/TEAD signaling and spheroid formation, invasion and migration, suggesting that SFN may target CD44v6 to attenuate YAP1/TEAD signaling and the cancer phenotype. This is consistent with the findings that CD44v6 knockdown attenuates YAP1/TEAD-dependent transcription and reduces YAP1/TEAD target gene (CCND1, CYR61) mRNA and protein levels.

Similar, but not identical, regulatory relationships have been observed in other cancer types. For example, SFN treatment reduces CD44v6 level in prostate cancer46, SFN reduces CD44 and ALDH1 in oral squamous cell carcinoma47, CD44 knockdown reduces YAP1 signaling and YAP1 target gene expression in lung cancer29, CD44/YAP1 cooperation has been described in hepatocellular carcinoma30 and CD44 modulates ERK1/2, Akt and YAP1 signaling31. Our present studies link SFN, CD44v6 and YAP1/TEAD in a single pathway.

Constitutively active YAP1 restores the cancer phenotype in CD44v6 deficient cells

To provide data confirming that the attenuated cancer phenotype in CD44v6 deficient cells requires loss of YAP1, we showed that expression of constitutively active YAP1 in these cells partially restores the cancer phenotype. We also created multiple CD44v6 knockout clonal cell lines and showed that these cells display reduced spheroid formation, invasion and migration and this was associated with reduced YAP1 and increased YAP1-P levels. This is an important finding, as reduced YAP1 activity is associated with cytoplasmic accumulation of phosphorylated YAP1 which is then degraded in the proteasome to terminate YAP1 signaling48. We also show that YAP1/TEAD transcription activity is reduced in all five CD44v6 knockout cell lines and that this is associated with reduced expression of YAP1/TEAD target proteins (cyclin D1 and CYR61).

CD44v6 loss is associated with reduce stem cell marker expression and reduced EMT

We also examined the impact of CD44v6 knockdown on stem cell (Sox2) and EMT (fibronectin, Twist, Slug, E-cadherin) markers. Like CD44v6, Sox2 is a stem cell marker protein49–51. We show a consistent loss of Sox2 in CD44v6 knockout cells suggesting reduced stemness. High levels of Twist, Slug and fibronectin and low levels of E-cadherin predict an aggressive cancer phenotype52. We show that CD44v6 knockout reduced Twist and increased E-cadherin indicating suppression of EMT.

SFN treatment suppresses YAP1 signaling, stemness and EMT in tumors and these changes are mimicked by CD44v6 knockdown

We wanted to confirm that the signaling response in tumors is consistent with our findings in cultured cells. We show that SFN treatment reduces tumor growth and YAP1 and pan-TEAD levels, and that this is associated with reduced YAP1/TEAD target protein (Cyclin D1, CYR61) levels, and reduced EMT (reduced Twist and Slug, increased E-cadherin). We also examined the impact of CD44v6 knockout on YAP1/TEAD signaling in tumors. CD44v6 knockout produced a dramatic reduction in tumor growth and YAP1/TEAD signaling, and this was associated with reduced cyclin D1 and CYR61 levels. Stemness (Sox2) and EMT (reduces Twist and fibronectin and increased E-cadherin) were also reduced.

These finding suggests that SFN treatment reduces CD44v6 level in cultured ECS cells and tumors which leads to reduced YAP1/TEAD signaling and reduced stemness and EMT. Based on these findings, we suggest that CD44v6 is important in maintaining YAP1/TEAD signaling to drive the CSCC cancer phenotype, and that SFN treatment reduces CD44v6 which attenuates YAP1/TEAD signaling to attenuate the cancer phenotype and tumor growth (Fig. 6F).

Acknowledgements

This work was supported by National Institutes Grants (R01 CA211909) to Richard L. Eckert and utilized the facilities of the Greenebaum Comprehensive Cancer Center (P30 CA134274) at the University of Maryland School of Medicine.

Support:

National Institutes of Health (R01 CA211909) to Richard L. Eckert and utilized the facilities of the Greenebaum Comprehensive Cancer Center (P30 CA134274) at the University of Maryland School of Medicine.

Abbreviations:

CYR61

Cysteine-rich angiogenic inducer 61

TEAD

Transcriptional enhancer factor

CCND1

cyclin D1

YAP

yes-associated protein

CTGF

connective tissue growth factor

SFN

Sulforaphane

Footnotes

Conflict of Interest

The authors declare no conflict of interest.

Data Sharing

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

References

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